The peptide hormone, insulin, regulates metabolism to homeostatically maintain blood glucose levels within a narrow physiological range. In pancreatic ss-cells, insulin is made and stored at high concentration within secretory granules. Physiological stimulation of insulin secretion (multiple times per day) requires very active synthesis of new insulin to replenish secretory granule reserves. Insulin synthesis begins with translation of preproinsulin for delivery into the lumen of the endoplasmic reticulum (ER). Therein, proinsulin must properly fold, which is easier than it sounds: proinsulin is a disulfide-challenged protein. Moreover, when beta cells are forced to synthesize higher levels of proinsulin than they are genetically-programmed to handle, they risk further proinsulin misfolding/disulfide mispairing, which leads to secretory pathway stress. The objective of this new grant cycle is to better understand proinsulin folding and export from the ER. We hypothesize that misfolding of a subfraction of proinsulin in the ER can block insulin production derived from the other subfracton of bystander proinsulin molecules, backlogging the protein in the ER, and driving ER stress, beta cell failure, loss of pancreatic insulin content, and diabetes. We propose four Specific Aims: 1) To elucidate the molecular mechanism(s) by which newly-described point mutations in the coding sequence of preproinsulin lead to human diabetes in neonates and adults. 2) To characterize ss-cell ER oxidoreductases. 3) To develop a new cell culture-based system to dissect steps leading to beta cell death. 4) To develop an in vivo analysis of pancreatic insulin production in diabetes.